The INSIG proteins are key regulators of sterol homeostasis in mammals, where they interact with the transmembrane domains of proteins with a Sterol Sensing Domain (SSD) and effect changes in the location or stability of the SSD clients. We recently discovered that the yeast S. cerevisiae has homologues of INSIGs, called Nsglp and Nsg2p, that similarly regulate the stability of yeast HMG-CoA reductase isozyme Hmg2p. Yeast Hmg2p has an SSD in its transmembrane region, which is required for the interaction and effects of yeast INSIGs on Hmg2p stability. Thus, the INSIG-SSD client interaction is broadly conserved allowing analysis of unknown features of INSIG action by use of facile yeast approaches to study the Nsglp- Hmg2p interaction as a model of all INSIG-SSD client interactions. The INSIG-SSD interaction in mammals requires sterols, and our preliminary evidence indicates that this aspect of the Nsg1p-Hmg2p interaction is similarly conserved. Taken together, these results indicate that the INSIG-SSD client interaction has been extant in biology for over 1 billion years, and its study in yeast will allow understanding of the mechanism and actions of the INSIG proteins in all cases. Specifically we plan to 1) Test the hypothesis that a late sterol pathway product regulates the interaction of the Nsg1p-Hmg2p pair. We will discover the identity of this regulator using a variety of approaches, and confirm its function using a number of in vitro assays of Nsg function. 2) Examine the sequence requirements for Nsg1p-Hmg2p interaction. We will test the role of the Hmg2p SSD in Nsglp- Hmg2p interaction, and similarly explore the role of conserved residues in the Nsg protein. We will also use yeast techniques to perform unbiased screens of these portions of the proteins to complement the directed tests of conserved motifs. In this way the details of an INSIG-SSD domain interaction will be delineated for the first time. Finally, 3) we will use a combination of proteomic techniques and phenotype-based genetic screens to explore other possible functions for the Nsglp protein. Because the INSIG proteins are so important in sterol regulation, any use of them as potential clinical targets will require a detailed understanding of they mechanisms and any possible involvement in unknown aspects of cell biology.